Characterization of Protein Cross-Links via Mass Spectrometry and an Open-Modification Search Strategy
ABSTRACT Protein−protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein−protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be “post-translationally” modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1−b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.
Article: Protein-protein interactions in the assembly and subcellular trafficking of the BACE (beta-site amyloid precursor protein-cleaving enzyme) complex of Alzheimer's disease.[show abstract] [hide abstract]
ABSTRACT: The correct assembly of the BACE (beta-site amyloid precursor protein-cleaving enzyme or beta-secretase) complex and its subsequent trafficking to cellular compartments where it associates with the APP (amyloid precursor protein) is essential for the production of Abeta (amyloid beta-peptide), the protein whose aggregation into senile plaques is thought to be responsible for the pathogenesis of AD (Alzheimer's disease). These processes rely upon both transient and permanent BACE-protein interactions. This review will discuss what is currently known about these BACE-protein interactions and how they may reveal novel therapeutic targets for the treatment of AD.Biochemical Society Transactions 12/2007; 35(Pt 5):974-9. · 3.71 Impact Factor
Article: Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides.[show abstract] [hide abstract]
ABSTRACT: Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes.Bioorganic & Medicinal Chemistry Letters 12/2003; 13(22):4023-6. · 2.55 Impact Factor
Article: Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.[show abstract] [hide abstract]
ABSTRACT: Mass spectrometry with chemical cross-linking was used to probe the conformational changes of HSA (human serum albumin) in solution on interaction with monounsaturated OA (oleic acid) or polyunsaturated AA (arachidonic acid) or DHA (docosahexaenoic acid). Fatty acid-free or -bound HSA was modified with lysine-specific cross-linkers and digested with trypsin. Cross-linked peptides were analysed by nano-electrospray ionization MS to localize the sites of cross-linking. Our data indicated that a local conformational change involving movement of the side chains of Lys-402 of subdomain IIIA or Lys-541 of subdomain IIIB occurred upon binding of all three fatty acids. Our data also indicated that the side chains of Lys-205 (IIA) and Lys-466 (IIIA) moved closer towards each other upon binding AA or DHA, but not OA, suggesting that the conformations of HSA when bound to mono- and poly-unsaturated fatty acids are distinctively different. While these observations agreed with previous X-ray crystallographic studies, the distances between epsilon-amino groups of most cross-linked lysine pairs were shorter than the crystal structure predicted, possibly reflecting a discrepancy between the solution and crystal structures. This method can serve as a useful complement to X-ray crystallography, particularly in probing the structure of a protein in solution.Biochemical Journal 06/2005; 387(Pt 3):695-702. · 4.90 Impact Factor